Urine-derived Epithelial Cells Research Articles

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

Urine-Derived Epithelial Cells as a New Model to Study Renal Metabolic Phenotypes of Patients with Glycogen Storage Disease 1a.

Glycogen storage diseases (GSDs) represent a model of pathological accumulation of glycogen disease in the kidney that, in animal models, results in nephropathy due to abnormal autophagy and mitochondrial function. Patients with Glycogen Storage Disease 1a (GSD1a) accumulate glycogen in the kidneys and suffer a disease resembling diabetic nephropathy that can progress to renal failure. In this study, we addressed whether urine-derived epithelial cells (URECs) from patients with GSD1a maintain their biological features, and whether they can be used as a model to study the renal and metabolic phenotypes of this genetic condition. Studies were performed on cells extracted from urine samples of GSD1a and healthy subjects. URECs were characterized after the fourth passage by transmission electron microscopy and immunofluorescence. Reactive oxygen species (ROS), at different glucose concentrations, were measured by fluorescent staining. We cultured URECs from three patients with GSD1a and three healthy controls. At the fourth passage, URECs from GSD1a patients maintained their massive glycogen content. GSD1a and control cells showed the ciliary structures of renal tubular epithelium and the expression of epithelial (E-cadherin) and renal tubular cells (aquaporin 1 and 2) markers. Moreover, URECs from both groups responded to changes in glucose concentrations by modulating ROS levels. GSD1a cells were featured by a specific response to the low glucose stimulus, which is the condition that more resembles the metabolic derangement of patients with GSD1a. Through this study, we demonstrated that URECs might represent a promising experimental model to study the molecular mechanisms leading to renal damage in GSD1a, due to pathological glycogen storage.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases.

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

Urine-Derived Epithelial Cell Lines: A New Tool to Model Fragile X Syndrome (FXS).

Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition associated with intellectual disability and behavioral problems due to the lack of the Fragile X mental retardation protein (FMRP), which plays a crucial role in synaptic plasticity and memory. A desirable in vitro cell model to study FXS would be one that can be generated by simple isolation and culture method from a collection of a non-invasive donor specimen. Currently, the various donor-specific cells can be isolated mainly from peripheral blood and skin biopsy. However, they are somewhat invasive methods for establishing cell lines from the primary subject material. In this study, we characterized a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). The urine-derived cells expressed epithelial cell surface markers via fluorescence-activated cell sorting (FACS). We observed inter, and the intra-tissue CGG mosaicism in the PBMCs and the urine-derived cells from participants with FXS potentially related to the observed variations in the phenotypic and clinical presentation FXS. We characterized these urine-derived epithelial cells for FMR1 mRNA and FMRP expression and observed some expression in the lines derived from full mutation mosaic participants. Further, FMRP expression was localized in the cytoplasm of the urine-derived epithelial cells of healthy controls. Deficient FMRP expression was also observed in mosaic males, while, as expected, no expression was observed in cells derived from participants with a hypermethylated full mutation.

Development of 3D human kidney model using Urine-Derived Epithelial Cells and HUVECs

Background and Hypothesis: Development of realistic, scaffold-free 3D organoids/tubuloids such as renal-like tissue, can serve as models for study of renal drug toxicity/screening, tissue repair, and renal disease. Previous studies utilized urine-derived epithelial cells (UDEpCs) to create viable tubuloid structures suspended in 3D Matrigel, displaying renal epithelial gene expression and functionality. Alternatively, renal proximal tubule epithelial cells (PTECs) with HUVECs were co-cultured in a monolayer to improve the model. Therefore, we hypothesized that the addition of HUVECs will help to establish and optimize growth conditions of UDEpCs in a 3D environment.
 Experimental design: UDEpCs were isolated and co-cultured with tdTomatoHUVECs in Matrigel with different combinations of growth media. Cell growth and endothelial cell tube formation were examined daily. PTECs and tdTomatoHUVECs were also used to make spheroids in low-binding plates. Real-time PCR analysis was performed to examine gene expression for epithelial markers (ABCC4, PAX8), an endothelial marker (PECAM1), and TGF-β in spheroids.
 Results: A single cell-derived colony was isolated from human urine and was double positive for CD10 and CD13, two renal PTEC markers. Co-culture of UDEpCs and tdTomato-HUVECs in Matrigel dramatically increased the growth, survival, and tubuloid formation of both cell types, for up to 12 days. Total RNA from spheroids showed a decline over time in all conditions. Gene expression was decreased in co-culture conditions, but patterns of gene expression were maintained.
 Conclusions and Potential Impact: Future studies will examine gene expression in Matrigel cells, further optimize conditions for growth of UDEpCs, and perform functional assays, such as brush border enzyme activity. Improved methods to develop 3D kidney organoids/tubuloids will produce more accurate models for renal function studies. Developing a functional model mimicking in vivo processes can lead to new applications for creating novel therapies and treatment strategies.

Generation of urine-derived induced pluripotent stem cells from a patient with phenylketonuria.

The aim of the study was to establish an induced pluripotent stem cell line from urine-derived cells (UiPSCs) from a patient with phenylketonuria (PKU) in order to provide a useful research tool with which to examine the pathology of this rare genetic metabolic disease. Urine-derived epithelial cells (UCs) from a 15-year-old male patient with PKU were isolated and reprogrammed with integration-free episomal vectors carrying an OCT4, SOX2, KLF4, and miR-302-367 cluster. PKU-UiPSCs were verified as correct using alkaline phosphatase staining. Pluripotency markers were detected with real-time PCR and flow cytometry. Promoter methylation in two pluripotent genes, NANOG and OCT4, was analyzed using bisulphite sequencing. An embryoid body (EB) formation assay was also performed. An induced pluripotent stem cell line (iPSC) was generated from epithelial cells in urine from a patient with PKU. This cell line had increased expression of stem cell biomarkers, it efficiently formed EBs, it stained positive for alkaline phosphatase (ALP), and it had a marked decrease in promoter methylation in the NANOG and OCT4 genes. The PKU-UiPSCs created here had typical characteristics and are suitable for further differentiation.

In vitro and in vivo differentiation of induced pluripotent stem cells generated from urine-derived cells into cardiomyocytes.

ABSTRACTGeneration of human cardiomyocytes from cells derived from various sources, including skin biopsy, has been made possible by breakthrough advances in stem cell research. However, it is attractive to build up a negligibly invasive way to create induced pluripotent stem (iPS) cells. In this study, we created iPS cells from human urine-derived epithelial cells by gene transduction using lentiviral vectors in a totally noninvasive manner. Then, we induced the differentiation of iPS cells into functional cardiomyocytes both in vitro and in vivo. Action potentials were recorded in putative cardiomyocytes and spontaneous beating cells were observed. Our results offered an alternative method to generate cardiomyocytes in a totally noninvasive manner from an easily accessible source. The availability of urine and its potent reprogramming characteristics will provide opportunities for the use of cells with specific genotypes to study the pathogenesis and molecular mechanisms of disease in vitro.

Tissue-specific differences in the proportion of mosaic large NF1 deletions are suggestive of a selective growth advantage of hematopoietic del(+/−) stem cells

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients.

Genetic heterogeneity of the human papovaviruses BK and JC.

We have examined the structure and infectivity of BKV and JCV genomes from prototype strains after cell culture passage and of BKV genomes from primary isolates. Genomic structures were determined by restriction endonuclease analysis of molecularly cloned DNA. Infectivity was determined by transfection of the cloned genomes into urine-derived epithelial cells and assaying for viral proteins and virus production. Prototype BKV DNA, which was cloned after 14 passages in three different cell lines, contained no alterations in restriction enzyme sites and was infectious. In contrast, prototype JCV acquired changes in the late region of the genome during passage in cell culture and the cloned DNA was not infectious. Urine-derived cells were used to isolate virus from the urine of two renal transplant patients and one asymptomatic individual. The genome of the virus isolated from the normal individual was indistinguishable from prototype BKV except for a 60-base pair deletion, which was localized between 0.62 and 0.72 map units. Two isolates from transplant patients differed from each other and from prototype BKV at a number of restriction enzyme cleavage sites located in the early region and were infectious. Genomes containing deletions from 100 to 600 base pairs were also cloned but were not infectious.

Propagation and primary isolation of papovavirus JC in epithelial cells derived from human urine.

Human papovavirus JC, previously passaged in amnion cells or in primary human fetal glial cells, replicated efficiently in urine-derived epithelial cells. Primary isolation of the virus from brain extracts was possible in urine-derived cells, but these cells were not as sensitive as primary human fetal glial cells for this purpose. Primary isolations of human papovavirus JC from urine sediments of renal transplant patients were made in urine-derived cells.